Laparoscopic versus traditional open splenectomy for hepatocellular carcinoma with hypersplenism / 华中科技大学学报（医学）（英德文版）

This study aimed to examine the efficacy of the laparoscopic vs. traditional open splenectomy for hepatocellular carcinoma (HCC) with hypersplenism. Between 2002 and 2013, 51 Chinese HCC patients with hypersplenism underwent either simultaneous laparoscopic splenectomy plus anticancer therapies (Lap-S&A) (n=25) or traditional open splenectomy plus anti-cancer therapies (TOS&A) (n=26). The outcomes were reviewed during and after the operation. Anti-cancer therapies for HCC included laparoscopic hepatectomy (LH) and laparoscopic microwave ablation (LMA). The results showed that there was no significant difference in the operating time between the two groups, but the blood loss and blood transfusion were less, pain intensity after surgery was weaker, the time to first bowel movement, time to the first flatus and postoperative hospital stay were shorter, and the postoperative complication rate and the readmission rate were lower in the Lap-S&A group than in the TO-S&A group. Two patients in the Lap-S&A group and one patient in the TO-S&A group died 30 days after surgery. However, no significant difference in the mortality rate was noted between the two groups. It was concluded that simultaneous Lap-S&A holds the advantages of more extensive indications, lower complication incidence and less operative expenditure than conventional open approach and it is a feasible and safe approach for HCC with hypersplenism.

Establishment of surfactant-associated protein A suicide gene system and analysis of its activity / 华中科技大学学报（医学）（英德文版）

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.

Establishment of surfactant-associated protein a suicide gene system and analysis of its activity / 华中科技大学学报（医学）（英德文版）

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.

Inactivation of Smad4 is a prognostic factor in intrahepatic cholangiocarcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Smad4 is found mutated in many cancers. It acts as a tumor suppressor in the regulation of TGF-β signaling pathway. The objective of this work was to study the expression of Smad4 in intrahepatic cholangiocarcinoma (ICC) and its relationship with the biological behavior and prognosis of the disease.</p><p><b>METHODS</b>Forty-nine paraffin-embedded ICC specimens and nine normal liver tissues were analyzed by immunohistochemical methods using Smad4 monoclonal antibodies. The expression of Smad4 was compared with the clinical pathological characteristics of the patients.</p><p><b>RESULTS</b>The expression of Smad4 was 100% positive in normal liver tissues, which was higher than that in the ICC (44.9%). Negative labeling of the Smad4 protein was found in 26.1% (6/23) of well-differentiated ICCs and 61.5% (16/26) of poorly to moderately differentiated ICCs, and 34.3% (12/35) and 71.4% (10/14) showed negative Smad4 labeling (P = 0.018) of ICC at pathological Tumor Node Metastasis (pTNM) stage I-II and pTNM stage III-IV separately. Furthermore, 72% (8/11) of lymph node metastatic ICCs and 73.3% (11/15) of intrahepatic metastatic ICCs showed negative labeling of the Smad4 protein. The loss of Smad4 expression in those metastatic ICCs was significantly more severe compared with non-metastatic ICCs (P = 0.000).</p><p><b>CONCLUSIONS</b>The expression of Smad4 was associated with the histological grade, clinical stage, and metastasis of ICC (P < 0.05). The detection of Smad4 may be helpful in determining the degree of malignancy and prognosis of ICC.</p>

Chloroquine relieves acute lung injury in rats with acute hemorrhagic necrotizing pancreatitis / 华中科技大学学报（医学）（英德文版）

This study preliminarily investigated the mechanism by which chloroquine (CQ) relieves acute lung injury (ALI) complicated in acute hemorrhagic necrotizing pancreatitis (AHNP). Sixty male Wistar rats were randomized into sham-operated group (group A, n=10), AHNP group (group B, n=10), L-arginine-treated group (group C, n=10), L-N-nitro-L-arginine methyl ester (NAME)-treated group (group D, n=10), CQ-treated group (group E, n=10) and CQ+L-NAME-treated group (group F, n=10). TLR4 expression was measured by using real time-PCR and Western blotting respectively. The results showed that, in the group B, the expression of TLR4 and the levels of TNF-α and IL-6 in the lungs were significantly increased, and the nitric oxide (NO) concentration was reduced, as compared with those in the group A (P<0.05 or P<0.01). Lung injury was aggravated with the increased expression of TLR4. When the inhibitor and stimulator of TLR4, namely L-Arg and L-NAME, were added respectively, lung injury was correspondingly relieved or aggravated (P<0.05 or P<0.01). In the group E, TLR4 expression was substantially lower and NO concentration higher than those in the group B (P<0.05 or P<0.01). However, in the group F, NO concentration was markedly decreased, and the inhibitory effect of CQ on TLR4 expression and the relief of lung injury were weakened when compared with those in the group E (P<0.05 or P<0.01). It was concluded that TLR4 may play an important role in the pathogenesis and development of ALI complicated in AHNP. CQ could relieve ALI by decreasing the TLR4 expression and increasing the NO release.

Chloroquine relieves acute lung injury in rats with acute hemorrhagic necrotizing pancreatitis / 华中科技大学学报（医学）（英德文版）

This study preliminarily investigated the mechanism by which chloroquine (CQ) relieves acute lung injury (ALI) complicated in acute hemorrhagic necrotizing pancreatitis (AHNP). Sixty male Wistar rats were randomized into sham-operated group (group A, n=10), AHNP group (group B, n=10), L-arginine-treated group (group C, n=10), L-N-nitro-L-arginine methyl ester (NAME)-treated group (group D, n=10), CQ-treated group (group E, n=10) and CQ+L-NAME-treated group (group F, n=10). TLR4 expression was measured by using real time-PCR and Western blotting respectively. The results showed that, in the group B, the expression of TLR4 and the levels of TNF-α and IL-6 in the lungs were significantly increased, and the nitric oxide (NO) concentration was reduced, as compared with those in the group A (P<0.05 or P<0.01). Lung injury was aggravated with the increased expression of TLR4. When the inhibitor and stimulator of TLR4, namely L-Arg and L-NAME, were added respectively, lung injury was correspondingly relieved or aggravated (P<0.05 or P<0.01). In the group E, TLR4 expression was substantially lower and NO concentration higher than those in the group B (P<0.05 or P<0.01). However, in the group F, NO concentration was markedly decreased, and the inhibitory effect of CQ on TLR4 expression and the relief of lung injury were weakened when compared with those in the group E (P<0.05 or P<0.01). It was concluded that TLR4 may play an important role in the pathogenesis and development of ALI complicated in AHNP. CQ could relieve ALI by decreasing the TLR4 expression and increasing the NO release.

Expression of MT1-MMP and its significance in rabbit VX2 tumor tissues after transarterial embolization with hydroxyapatite nanoparticles / 中华外科杂志

<p><b>OBJECTIVE</b>To study location of MT1-MMP and effect of its change in expression on rabbit VX2 tumor tissues after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol.</p><p><b>METHODS</b>Sixty rabbits implanted with tumor tissue of cell line VX2 were divided into three groups (control group, lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group). The transarterial embolization was performed super-selectively via gastro- duodenal artery of rabbits, each rabbit in control group was inserted with 1 ml normal saline,that in lipiodol group was inserted with 0.3 lipiodol ml/kg, also 0.3 ml hydroxyapatite nanoparticles loaded with lipiodol per kg for that in the last group. Results of embolization were detected by using CT scanning 3 days after operation. After two weeks, all tumors were took out as specimens to investigate location of MT1-MMP in VX2 tumor tissues,and also to determine the change of its expression in tumor tissues after embolization with different medicines, with three-step immunohistochemical technique (S-P). MT1-MMP mRNA was measured by RT-PCR to determine whether there were differences in three groups. Western blot technique was performed to determine difference of MT1-MMP protein expression of in three groups.</p><p><b>RESULTS</b>Immunohistochemical results exposed that MT1-MMP was expressed on membrane of tumor cells and in extracellular matrix of tumor cells. Comparison of MT1-MMP expression in control group with that in other two groups, showed a significant lower level in control group (P < 0.05). There was no difference in MT1-MMP expression between lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group (P > 0.05). Western blot supported this conclusion. RT-PCR detecting MT1-MMP mRNA was found no differences among three groups (P > 0.05).</p><p><b>CONCLUSIONS</b>MT1-MMP was mainly expressed on membrane of tumor cells and in extracellular matrix of tumor cells. There was an increasing tendency on expression of MT1-MMP in tumor tissues and extracellular matrix after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol,it might be one of important mechanisms provoking high recurrence rate for hepatocellular carcinoma after treatment embolization.</p>

Research of signaling pathway of vascular endothelial growth factor regulating by hepatitis B virus X protein / 中华外科杂志

<p><b>OBJECTIVE</b>To study the function of nuclear factor-kappaB (NF-kappaB) signaling pathway in regulating vascular endothelial growth factor (VEGF) by hepatitis B virus X protein (HBx).</p><p><b>METHODS</b>After the establishment of L02-HBx cell line with stable transfected HBx gene, NF-kappaB signaling pathway blocker PDTC was introduced to cut off its signal transduction. Double immunofluorescent staining and laser scanning confocal microscopy were applied to study the activation and deactivation of NF-kappaB signaling pathway. Real-time PCR and Western blot were used to observe the expression of VEGF gene before and after the HBx transfection, as well as the treatment with PDTC.</p><p><b>RESULTS</b>The NF-kappaB signaling pathway of L02-HBx cells was activated after transfection with HBx gene as compared to the control L02 cells without treatment. The mRNA and protein levels of VEGF in L02-HBx cells increased 4.07 +/- 0.31 and 4.34 +/- 0.64 times respectively. The difference was of statistical significance (P < 0.05) in comparison with the control cells. The mRNA levels of VEGF decreased to 2.33 +/- 0.22 and 1.86 +/- 0.18(P < 0.05) and at the same time the expression of VEGF also reduced to 2.52 +/- 0.29 and 2.17 +/- 0.34 (P < 0.05), after treatment with 25.0 micromol/L and 50.0 micromol/L PDTC for 24 h respectively when the NF-kappaB signaling pathway was blocked. There was no significant difference in VEGF mRNA and protein levels when treated with 12.5 micromol/L PDTC for 24 h.</p><p><b>CONCLUSION</b>NF-kappaB signaling pathway maybe one of the routes through which HBx up-regulate the expression of VEGF to promote angiogenesis in hepatocellular carcinoma.</p>

Expressions of ATP binding cassette transporter genes in rat hepatic oval cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Liver regeneration occurs through hepatocytes after acute liver injury. However, severe liver injury activates bipotential oval cells from canals of Hering which can differentiate into hepatocytes and biliary epithelial cells. Most models of oval cell activation have employed potential carcinogens to inhibit hepatocyte replication in the face of a regenerative stimulus. Oval cells must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette transporters are cytoprotective efflux pumps that may contribute to the protection of these cells. The aim of this study was to determine the ABC transporter expressions in hepatic oval cells.</p><p><b>METHODS</b>A rat model was established by feeding 2-acetylaminofluorene combined with partial hepatectomy to activate hepatic oval cells. Oval cells were isolated and purified using selective enzymatic digestion and density gradient centrifugation from the heterogeneous hepatic cell population. The expressions of ABC transporter gene, including MDR1, MRP1 and Bcrp1, in isolated hepatic oval cells and hepatocytes were measured by quantitative real-time reverse transcription-polymerase chain reaction and those in rat liver tissues were measured by immunohistochemistry.</p><p><b>RESULTS</b>Compared to those in the rat hepatocytes, mRNA expressions of the genes encoding MDR1, MRP1 and Bcrp1 were increased up to 9-, 1.5- and 13.8-folds in hepatic oval cells. Immunohistochemical staining of rat liver slides demonstrated that the expression of MDR1 proteins was found around periportal areas, and Bcrp1 protein was found located on cell membranes.</p><p><b>CONCLUSION</b>Hepatic oval cells express high levels of the ABC transporter gene that may have cytoprotective functions during severe hepatotoxicity.</p>

Inducible expression of hypoxia-inducible factor 1alpha on the proliferation and invasion property of HepG2 cells under normoxia in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To study the inducible expression of hypoxia-inducible factor 1alpha (HIF-1alpha) on the proliferation and invasion property of HepG2 cells under normoxia in vitro.</p><p><b>METHODS</b>Constructed the HepG2(Tet-on-HIF-1alpha) cell line which could induce the expression of HIF-1alpha by doxycycline; Under normoxia in vitro, MTT assay was used to observe the proliferative and adhesive activity of cells, and the invasive activity was determined by transwell cell culture chamber method.</p><p><b>RESULTS</b>Under normoxia, the HIF-1alpha mRNA and protein of HepG2(Tet-on-HIF-1alpha) cells could be induced up to (5.899 +/- 2.176) and (2.179 +/- 0.742) folds by doxycycline (1 microg/ml); There were no difference of A(490 nm) between the Dox(+)and Dox(-) group in experiment detecting the proliferation activity (P > 0.05); But in adhesive experiment, the A(490 nm) of Dox (+) group was 0.662 +/- 0.058, higher than the Dox(-) group 0.526 +/- 0.808 (P = 0.008); The invasive cell number of Dox(+) group was 37.611 +/- 8.424, but in the Dox(-) group, the number was 25.333 +/- 8.117 (P < 0.01).</p><p><b>CONCLUSIONS</b>Under normoxia in vitro, the Tet-on gene regulate system could increase the HIF-1alpha protein by inducing the HIF-1alpha mRNA; HIF-1alpha has no influence with the proliferation activity, but it could enhance the adhesive and invasive properties of HepG2 cells.</p>

TNF-alpha induced reversal of multidrug resistance in human hepatocellular carcinoma cells / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the reversal of multidrug resistance in the cell line HepG2/ADM induced by TNF-alpha.</p><p><b>METHODS</b>HepG2/ADM cells were incubated with different concentrations of TNF-alpha (100, 500 and 2500 U/ml) for 72 h. Real-time PCR was performed to compare the mRNA levels of MDR1 with PPAR-alpha in the different concentrations of TNF-alpha treated cells. The Annexin V assay was used to check cell apoptosis induced by 0.5 mg/L adriamycin. Rhodamine 123 efflux assay and MTT assay were used to study P-gp activity and drug resistance in each group, respectively.</p><p><b>RESULTS</b>TNF-alpha could induce down-regulation of MDR1 and up-regulation of PPAR-alpha. Meanwhile, it could enhance cell cytotoxicity and cell apoptosis induced by 0.5 mg/L adriamycin.</p><p><b>CONCLUSIONS</b>TNF-alpha could partially reverse the multidrug resistance of HepG2/ADM cells by down-regulating the expression of MDR1 and up-regulating the expression of PPAR-alpha.</p>

The role of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance of hepatocellular carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To elucidate intracellular signal pathway in formation of multidrug resistance (MDR) of hepatocellular carcinoma (HCC) induced by its microenvironment, and to explore the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.</p><p><b>METHODS</b>Activity of ERK/MAPK was examined by Western blot technique through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein in HepG2 cells exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX. After being treated by the specific ERK/MAPK pathway inhibitor U0126, Western blot technique was used to analyze the alterations of the expression of P-gp, MRP1, LRP and HIF-1alpha at protein level. RT-PCR was used to analyze the alterations of the expression of HIF-1alpha mRNA. Cellular location of HIF-1alpha protein was determined by immunocytochemistry after being treated by U0126.</p><p><b>RESULTS</b>The activations of ERK/MAPK determined by the ratio of phosphorylated ERK/MAPK to the total ERK/MAPK were increased in varying degrees in HepG2 cells respectively exposed to different microenvironment. After being treated by U0126 for 12 h, the expressions of mdr1, MRP1, LRP genes and protein in those cells were decreased to some extent. However, the gene expression of HIF-1alpha was not influenced and only its protein was decreased. HIF-1alpha protein was reversely translocated into cytoplasm from nucleus after being treated by U0126.</p><p><b>CONCLUSIONS</b>ERK/MAPK pathway is involved in the course of the formation of MDR of HCC induced by microenvironment.</p>

Hepatic progenitor cell activation and expansion in human liver cirrhosis / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To confirm whether human hepatic progenitor cells (HPCs) occur in human liver cirrhosis, to investigate the relationship between the degree of HPCs activation and the degree of liver inflammation and to provide evidence that HPCs can differentiate into hepatocytes.</p><p><b>METHODS</b>Surgical specimens from 30 human cirrhotic livers and from 3 normal livers were investigated by light microscopy and immunohistochemical staining for CK7 (a marker of biliary differentiation) and SMA (a marker of hepatic stellate cell activation). The degree of portal inflammation was determined on routine stained sections. The number of HPCs and intermediate hepatocytes and the extent of the ductular reaction were assessed.</p><p><b>RESULTS</b>HPCs and ductular reaction were not observed in the normal livers. In liver cirrhosis cases the HPCs originated from the portal areas. With the increase of portal inflammation, HPCs and ductular reaction extending from the periphery of the liver cirrhosis nodules to the liver parenchyma and the intermediate hepatocyte proliferation were observed. The notable hepatic stellate cell activation occurred around the HPCs and ductular reaction. The number of HPCs and the extent of ductular reaction increased significantly as the portal inflammation increased. There were significant correlations between the number of HPCs with the number of intermediate hepatocytes. In addition, there was a strong correlation between the ALT and AST with the number of HPCs and intermediate hepatocytes.</p><p><b>CONCLUSION</b>Human hepatic progenitor cell activation exists in human liver cirrhosis. The inflammation is a trigger for HPCs activation. HPCs migration from the portal area to liver parenchyma and their differentiation into hepatocytes are important pathways for liver regeneration.</p>

Study on the mechanism of reversing multidrug resistance of hepatocarcinoma by bromocriptine / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of reversing multidrug resistance of hepatocarcinoma by bromocriptine (BCT) in vitro and in vivo.</p><p><b>METHODS</b>Three groups of cultured HepG2 cells were used: HepG2 (group A), the multidrug resistance HepG2/ADM cells (group B), and the HepG2/ADM cells treated with BCT (group C). Rhodamine 123 test was used to detect the function of P-gp protein. MTT assay was performed to examine the IC50. Multidrug resistance index to common five anticancer drugs of different concentrations of BCT and immunocytochemistry was performed to detect the expression of PKC-a protein. P-gp protein levels of the cells of each group were determined by Western blot. In addition, HepG2 and HepG2/ADM were injected into the livers of the nude mice (NM) (named NM HepG2, NM ADM, NM BCT groups respectively). The BCT group mice were treated with bromocriptine through gastric feedings. The sizes of the tumor growths in the livers were measured using B ultrasound. The MDR1mRNA levels in these tumor tissues were determined by reverse transcription polymerase chain reaction (RT-PCR) and the apoptosis rates of them were measured with TUNEL assay. 99mTc-MIBI SPECT was performed to detect the tumor 99mTc-MIBI accumulation index before and after the BCT treatment.</p><p><b>RESULTS</b>The rate of reversing resistance to ADM by BCT was 45.68% shown by using MTT assay, and the intracellular Rho123 accumulation increased more than two times compared with the control group shown by flow cytometric assay at the concentration of 10 micromol/L BCT and the effect was time-dependent. Between group B and group C there was a significant difference in the expression of PKC-alpha protein by immunocytochemistry detection (q = 5.37, P < 0.01), but there was no significant difference in the expression of P-gp protein between the two groups (q = 1.86, P > 0.05) . There was no notable difference of growth rates of the transplanted liver tumors among the three NM groups (F = 6.39, P > 0.05), and the inhibition rate of tumor volume and weight in NM BCT was higher than that in NM ADM (q1 = 5.89, q2 = 4.92, P < 0.01), but similar to that in NM HepG2 (q1 = 2.47, q2 = 3.02, P > 0.05). No difference was detected in MDR1mRNA between NM ADM and NM BCT using RT-PCR (q = 3.71, P > 0.05). The average number of apoptotic cells per high-power field (25.7+/-1.8) in NM BCT tumor tissues was higher than that in group ADM (2.7+/-0.2) (q = 3.72, P < 0.01), but similar to that of NM HepG2 (23.9+/-1.6) (q = 1.43, P > 0.05). The uptake of 99mTc-MIBI in all the NM after BCT therapy was significantly higher than that before the BCT treatment (t = 3.58, P < 0.01).</p><p><b>CONCLUSIONS</b>BCT can reverse multidrug resistance of hepatocarcinomas by inhibiting the function of P-gp protein and can enhance the susceptibility of HepG2/ADM cells to cytotoxic drugs.</p>

Hypoxia-inducible factor-1 alpha dependent expression and significance of the related multidrug resistance genes induced by hypoxia in human hepatocarcinoma cell / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the mechanism of multidrug resistance of hepatocellular carcinoma induced by hypoxia and the potential role of hypoxia-inducible factor-1 alpha (HIF-1 alpha) and multidrug resistance related genes.</p><p><b>METHODS</b>Human hepatocarcinoma cell lines HepG2 cells were exposed to hypoxia and were transfected by plasmid HIF-1 alpha/PCDNA3, respectively. The expressions of multidrug resistance gene (mdr1), multidrug resistance protein (MRP1), and lung resistance protein (LRP) gene at the mRNA and the protein levels in the above two groups were respectively analyzed by real-time fluorescent quantitative PCR and Western-blot technique.</p><p><b>RESULTS</b>In the hypoxia group, the expressions of mdr1, MRP1 and LRP were stepped up correlating to the degree of hypoxia, especially the prominent increase in the expression of MRP1. Furthermore, they were synchronous with the changes of the expression of HIF-1 alpha. Also the increased expression of mdr1, MRP1, and LRP gene was observed in transfected HepG2 cells by plasmid HIF-1 alpha/PCDNA3.</p><p><b>CONCLUSIONS</b>Resistance of hepatocellular carcinoma to chemotherapeutics could be induced by hypoxia. HIF-1 alpha may be critical to the upregulation of the expression of the related multidrug resistance genes induced by hypoxia. HIF-1 alpha and these related multidrug resistance genes could be potential molecular targets for reversing multidrug resistance of hepatocellular carcinoma.</p>

Tumor necrosis factor related apoptosis-inducing ligand induces apoptosis and sensitizes resistant hepatic cancer cells to chemotherapy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effective of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) combination with chemotherapeutic agent inducing apoptosis in resistant hepatic cancer cells.</p><p><b>METHODS</b>HepG2 cells resistant to Adriamycin (HepG2/ADR) were induced stepwise. The effects of TRAIL in combination with ADM (0.1 mg/L) on promoting apoptosis in HepG2/ADR were analyzed, the proliferation was observed by MTT assay, the apoptosis of cells was also observed by flow cytometry and TUNEL method.</p><p><b>RESULTS</b>HepG2/ADR was confirmed resisting to ADM. Treated with TRAIL combination with ADM (0.1 mg/L), it showed significant inhibitory effect on the growth of HepG2/ADM, the percentage of apoptosis was increased as comparison with control at 24 h.</p><p><b>CONCLUSION</b>MDR1 might not take part in resistance to TRAIL-induced apoptosis. TRAIL dramatically augmented the sensitivity to chemotherapeutic agents in HepG2/ADR. Combined TRAIL with chemotherapeutic agents treatment could be a novel and attractive strategy to drug-resistant/ TRAIL-resistant tumor cells.</p>

HBx gene facilitates the proliferation activity of hepatoma cells in vitro and in vivo / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of HBx gene on proliferation activity of hepatoma cells in vitro and in vivo.</p><p><b>METHODS</b>The plasmid pHA-HBx carrying HBx gene was transfected into HepG(2) cells, and the positive clones were screened and identified with G418 and RT-PCR, respectively. The growth curve and population doubling time were calculated, and the cell cycle was analyzed by flow cytometry (FCM). The proliferation activity of transformed cells was measured with (3)H-TdR incorporation rate and nude mice model in vitro and in vivo.</p><p><b>RESULTS</b>The result of RT-PCR indicated that HBx gene was integrated into the genome DNA of HepG(2) cells and transcripted. The growth curve and population doubling time showed a high proliferation activity of transformed cells. The amount of cells at stage S and G(2)/M were significantly higher, and cells at stage G(0)/G(1) were lower than those in control group. The tumors developed from transfected cells grew much quicker than those developed from HepG(2) cells in nude mice model.</p><p><b>CONCLUSION</b>HBx gene can facilitate the proliferation of hepatoma cells both in vitro and in vivo.</p>

Interleukin-12 enhanced tumor necrosis factor related apoptosis-inducing ligand TRAIL-induced apoptosis in human hepatocellular carcinoma by inhibiting expression of survivin / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate therapeutic potential of TRAIL in hepatocellular carcinoma (HCC) and the mechanism of sTRAIL resistance and to reverse the resistance to sTRAIL-inducing apoptosis.</p><p><b>METHODS</b>The expression profiles of TRAILR were determined 60 HCC samples, in 20 normal liver tissues and 2 HCC cell lines HepG2 and SMMC-7721 by in situ hybridization. Cellular effects of sTRAIL in promoting apoptosis on HCC cell lines HepG2 and SMMC-7721 were analyzed after exposure to recombinant protein and after transfection with a cDNA expression construct. In vivo effects of sTRAIL on tumor growth were investigated using a nude mice HCC model of hepG2. Furthermore, the expression of survivin in HCC was detected, and treatment with antisence oligonucleotide was accepted. Finally, therapeutic effect on HCC by combining sTRAIL and interleukin-12 (IL-12) was detected.</p><p><b>RESULTS</b>Both DR4 and DR5 were present in all HCC tissues as well as normal hepatic tissues. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. Recombinant sTRAIL alone was found to have a slight activity as it killed a maximum of 15% of HCC cells within 24 h while killing over 70% of Jurkat cells. In vivo administration of the TRAIL gene couldn't inhibit tumor growth in a nude mice HCC model. Mostly, HCC tissue and both HCC cell lines expressed survivin, whereas normal liver tissue did not express survivin. Treatment with antisence oligonucleotide enhanced sTRAIL-inducing apoptosis. IL-12 significantly augmented sTRAIL-inducing apoptosis and inhibited survivin expression.</p><p><b>CONCLUSIONS</b>HCC cells are insensitive towards TRAIL-mediated apoptosis. Survivin may play a role in resistance to TRAIL-induced apoptosis in HCC, and antisence oligonucleotide could partly reverse the resistance to TRAIL-inducing apoptosis. IL-12 may sensitize HCC cells to TRAIL-induced apoptosis by preventing survivin. Combining gene therapy strategy such as combining gene therapy of TRAIL with IL-12 may be a promising maneuver to HCC.</p>

Hepatitis B x protein activated vascular endothelial growth factor expression through hypoxia inducible factor-1 pathway / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate whether hepatitis B x protein (HBx) stimulates vascular endothelial growth factor (VEGF) through hypoxia inducible factor-1 (HIF-1 alpha) pathway.</p><p><b>METHODS</b>Two plasmids including pIRES-EGFP-HBx and pTK-Hyg were co-transfected to a hepatocellular carcinoma cell line SMMC-7721. With fluorescence-positive and fluorescence-negative hygromycin-resistant colonies selected, expressions of VEGF and HIF-1 alpha in protein or/and mRNA level were detected.</p><p><b>RESULTS</b>Fluorescence-positive cells were stably integrated with HBx, in which expression of HIF-1 alpha and VEGF were upregulated. Fluorescence-negative cells did not express HBx, VEGF or HIF-1 alpha.</p><p><b>CONCLUSION</b>HBx can activate VEGF through HIF-1 alpha pathway.</p>

Antitumor effects of soluble TRAIL in human hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate therapeutic potential of soluble TRAIL (sTRAIL) in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Expression of TRAILR was determined by in situ hybridization in 60 samples of resected hepatocellular carcinoma, 20 samples of normal liver tissue near the margin of benign tumor and 2 HCC cell lines of HepG2 and SMMC-7721. The clinical data of the patients were analyzed as well as cellular effects of sTRAIL in promoting apoptosis on HCC cell lines HepG2 and SMMC-7721 (p53 gene mutated) after exposure to different concentrations of recombinant protein.</p><p><b>RESULTS</b>High death receptor (DR) expression and low DcR expression in HCC tissue differed from low DR expression and high DcR expression in the normal hepatic tissue with statistical significance. DR5, DR4, and DcR2 but not DcR1 were expressed in both cell lines. The expression of DR was closely correlated with HCC differentiation, with the weak expression in poor differentiation. The positive rate of DR expression in 32 cases of grade III-IV was significantly lower than that in 28 cases of grade I-II (P < 0.05). Cell apoptosis rates were 10%, 70% and 50% of HCC cells, Jurkat cells and human cholangiocarcinoma cell line QBC939 24 h after recombinant of TRAIL alone.</p><p><b>CONCLUSION</b>TRAILR expression is prevalent in HCC, with different receptor types existing. HCC is resistant to TRAIL-mediated apoptosis. The treatment of TRAIL alone only has a limited effect on inducing apoptosis on HCC cell lines of HepG2 and SMMC-7721.</p>